Pharmacology_ Toxicokinetics

1. TOXICOKINETICS
Mr Dilip N Kawane
M Pharm (Pharmacology)

2.  Toxicokinetics(TK) is defined by the ICH as ‘the generation of
pharmacokinetic data, either as an integral component in the conduct of
non-clinical toxicity studies or in specially designed supportive studies,
to assess systemic exposure.
 While developing a molecule as a therapeutic agent researchers consider
not only benefit but also risk associated with it.
 Hence toxicological evaluation got more importance in drug development
stages especially in preclinical stage.
Theoretical approach
development of pharmacokinetic models
that predict drug disposition after drug
administration. (Mathematics and
computer Techniques are heavily
utilized).
Experimental approach
development of biologic sampling
techniques, analytical methods for
the measurement of drugs and
metabolites, procedures that facilitate
data collection and manipulation.
The study of Toxicokinetics

3. Toxicokinetics (TK) is generation of kinetic data for systemic exposure
and toxicity assessment of the drug.
These studies help us to estimate the observed toxicity to that dose.
TK evaluation is very important in drug development phase in both
regulatory and scientific perspective.
There are several guidelines to conduct TK study in animals
recommended by regulatory bodies (OECD).
Toxicokinetics evaluation is useful in
 Selection of dose
 Dosing form
 Alternative dosing route
 Evaluation of toxicological mechanism
 Reduces the animal number
 Also used for the setting safe dose level in clinical phases

4. On the other hand, TK data are practically used for the purpose of drug
discovery such as lead-optimization and candidate-selection.
The primary objective of the toxicokinetics studies is,
• To describe the systemic exposure achieved in animals and its
relationship to dose level and the time course of the toxicity study .
• Exposure data in animals should be evaluated before human clinical
trials.
• Choice of species and treatment regimen used in non clinical studies .
• Information on systemic exposure of animals during repeated-dose
toxicity studies is essential for the interpretation of study results, to the
design of subsequent studies and to the human safety assessment.

5. A.Toxicokinetics
i) Absorption
• Transfer of chemicals from the gut lumen, lungs, or skin into the
general circulation involves movement across cell membranes, and
simple passive diffusion of the unionized molecule, down a
concentration gradient is the most important mechanism.
Absorption is the process by which the chemical enters the body. It
depends on the route of administration.
• Oral route – the GIT is the most important route of absorption, as
most acute poisonings involve ingestions.
• Dermal route – lipid solubility of a substance is an important factor
affecting the degree of absorption through the skin.

6. • Inhalational route – toxic fumes, particulate and noxious gases
may be absorbed through the lungs.
Bioavailability
• is the fraction of unchanged drug reaching the systemic
circulation following of non-vascular administration. Therefore, a
portion of the chemical fails to reach the systemic circulation in
original form after oral administration.
Extent of absorption
• It is often useful to determine how much of the toxicant actually
penetrates the membrane barriers (e.g skin or GIT) and gets into the
blood stream.
• The area under the curve (AUC) of the concentration- time
profiles for oral or dermal routes is compared with the AUC for IV
ROA.

7. ii) Distribution
• Distribution-is defined as the apparent volume into which
a substance is distributed. Volume of distribution (Vd) is
calculated from the dose taken and the resulting plasma
concentration:
Vd = dose /plasma concentration
• The importance of volume of distribution in toxicology is,
- Predicting peak blood concentration of the chemical taken
- Calculating the amount of substance in the body to verify the
quantity ingested
- Deciding whether to apply systemic toxin elimination technique.

8. • Factors determining the rate of distribution of chemicals in the
body are
I. Protein binding – chemicals highly bound to protein have
small volume of distribution
II. Plasma concentration – when the volume of distribution of
chemicals is small, most of the chemical remains in the plasma
III. Physiological barriers – chemicals will not uniformly
distributed to the body due to specialized barriers e.g blood
brain barrier.

9. iii) Biotransformation (metabolism)
•
Biotransformation is the biochemical transformation of a
chemical.
• It is a process by which the body transforms a chemical and
makes it more water soluble so the chemical can be eliminated
more rapidly via the kidney into the urine.
• Biotransformation can produce metabolites that are
pharmacologically active and toxic.
E.g. parathion→ parathoxon (toxic metabolite). Liver is the major
site of biotransformation for many chemicals & other organs
that are involved are lungs, kidneys, skin &so on. Interactions
during biotransformation include

10. • There are two phases of biotransformation
• Phase I – the drug is converted into more polar compound
e.g oxidation, reduction, &hydrolysis
• Phase II (conjugation) – a drug or its metabolite is
conjugated with an endogenous substance
e.g glucuronide conjugate
• Enzyme inhibition- by this the biotransformation of drugs is
delayed & is a cause of increased toxicity
• Enzyme induction- by this the biotransformation of drugs is
accelerated & is a cause of therapeutic failure.

11. • First – pass effect – is the biotransformation of some chemicals
by the liver during the initial pass from the portal circulation after
oral administration.
• Half life (t ½) –is the time required to reduce the blood
concentration of the chemical to half.

12. IV) Excretion
- Excretion is the final means of chemical elimination, either as
metabolites or unchanged parent chemical.
- Excretion through the lungs is the major route for gaseous
substances; and in the case of non-volatile water – soluble drugs, the
kidneys are the most important routes of excretion.
- Additional routes include sweat, saliva, tears, nasal secretions,
milk, bile and feces
- Excretion is a major determinant of the potential toxicity of a
foreign chemical, rapid excretion → less toxicity

13. • The kidney is a major route of excretion and it functions to
excrete body wastes and toxic chemicals.
• Many chemicals have to be biotransformed to more water-
soluble products before they can be excreted in the urine.
• Kidney damage (chemicals, infections, age)→ reduced
excretion-----------toxicity.

14. • Clearance – elimination of chemicals from the body may
be described by the term clearance (CL).
It is a quantitative measure of the volume of blood cleared of drug
per unit time,usually expressed in milliliter per 4 minute.
• Clearance is calculated as follows
CL = 0.7 (VD)/ (t1/2) = ml/min
Where the VD is expressed in milliliter per kilogram & the
half-life is expressed in minutes or hours.
Repeated exposure--- -------- accumulation
(increased toxicity)
(Kidney damage, liver
damage, infants, elderly)

15. • Certain points regarding the toxicokinetics of toxic agents;
Drug absorption after a toxic ingestion may be delayed and
prolonged;
The half-life and total body clearance are often lengthened;
Liver-metabolizing enzymes may become saturated, slowing
hepatic elimination;
Chemicals with large volumes of distribution are often highly
tissue-bound and measures to enhance their elimination are not
effective;
Poor perfusion of the liver and kidneys secondary to the toxic
effects of the substance may slow clearance.

16. IMPORTANCE OF TOXICOKINETICS:
 Toxicokinetics evaluation is important in drug development
stages.
TK data is important to know the toxic response(s) to that of drug
exposure obtained in drug development stages (preclinical).
 To set safe dose for clinical use of new drugs.
 Useful in the understanding of differences in responses or
sensitivity between individual animals, genders, species or life
stages, and supporting the extrapolation of findings in
experimental animals to humans.

17. Kinetic data can also support mode-of action analysis and
extrapolation across exposure routes.
Now a day toxicokinetics used in other areas also with other
areas of pharmacokinetics.
Measuring drug levels in non plasma samples (tissues, urine and
bile).
It helps in optimizing the dosage regimen for individual
patients.
It helps in explaining the drug interactions.
It helps in determining the influence of altered physiology on
drug ADME.

18. Importance of toxicokinetics
Drug Development
Formulation Development
Deciding Dosage Regimen
Designing Rational Dose, Frequency and Duration
Rational Drug Design (QSPKR)
Clinical Pharmacy
ADME study, Bioavailability and Bioequivalence studies
In Vitro –In Vivo correlation studies
Pharmacokinetics, Pharmacodynamics Relationship

19. Importance of saturation kinetics
Drugs that demonstrate saturation kinetics usually show the
following characteristics:
1. Elimination of drug does not follow simple first-order kinetics
that is, elimination kinetics are nonlinear.
2. The elimination half-life changes as dose is increased. Usually,
the elimination half-life increases with increased dose due to
saturation of an enzyme system.
3. The area under the curve (AUC) is not proportional to the amount
of bioavailable drug.
4. The saturation of capacity-limited processes may be affected by
other drugs that require the same enzyme or carrier-mediated system
(ie,competition effects).
5. The composition and/or ratio of the metabolites of a drug may be
affected by a change in the dose.

20. ALTERNATIVE METHOD TO ANIMAL TOXICITY TESTING
Alternative test methods are test methods that reduce, refine and
replace animal use.
 Reduction, refinement, and replacement are commonly referred
to as
 “the 3Rs (4Rs includes Rehabilitation) of alternatives”.
 The concept of 3Rs was first proposed by William Russell and
Rex Burch in the “Principles of Humane Experimental
Technique”.

21.  Alternatives to animal testing are the development and
implementation of test methods that avoid the use of live animals.
 Human biochemistry, physiology, pharmacology, and
endocrinology and toxicology has been derived from animal
models.
 10 to 100 millions of animals are using for experimentation in a
year
 Animals used experimentation distributed among zebra-fish to
Primates
 Vast majority of animals are sacrificed at end of research
programme

22. 1R= Replacement of test animals
2R=Refinement ( better tests)
3R=Reduction ( decrease of the number of animals)
4R=Responsibility ( tests scientifically acceptable)

23. a. Using of living system
In vitro Techniques
Invertebrates Animals
Micro-organisms
b. Using Non-living systems
Chemical Technique
Physical/Mechanical Systems
c. Use of computer simulation
These principles are now followed in many testing establishments
worldwide.
A. Replacement
Refers to the preferred use of non-animal methods over
animal methods whenever it is possible to achieve the same
scientific aim.

24. B. Reduction
Refers to methods that enable researchers to obtain
comparable levels of information from fewer animals, or to obtain
more information from the same number of animals.
A. Animal Sharing
B. Improved Statistical
C. Phylogenetic Reduction
D. Better Quality Animal
E. Post-Marketing Surveillance
F. Autopsies and Biopsies
G. Epidemiological Studies

25. C. Refinement refers to methods that alleviate or minimize
potential pain, suffering, or distress, and enhance animal welfare
for the animals used.
a) Decreased Invasiveness
b) Improved Instrumentation
c) Improved Control Pain
d) Improved Control of techniques

26. Globalization of R- concept
Dr. David B. Morton, expanded this concept to the Fifteen R's :-
1. Reduce the number used.
2. Refine end points and procedures.
3. Replace with in vitro, ex vitro methods when possible.
4. Respect all animals regardless of species.
5. Recognize any adverse effects.
6. Relieve pain with analgesics, distress with anxiolytics.
7. Refuse to carry out some procedures if concerned.
8. Reconsider protocol if unsure.
9. Read about the science and the ethical issues.
10.Reflect on the work you have carried out.
11.Reason out why you are doing the research
12.Record all your observations carefully.
13.Reward rather than cause harm and for happiness.
14.Reappraise techniques for efficacy.
15.Resolve to learn new techniques.

27. ACCORDING TO OECD
1. Complete replacement of the animal experiment
• TG 428 Skin absorption: in vitro method
• TG 430 In vitro skin corrosion: transcutaneous electrical
resistance (TER)
• TG 431 In vitro skin corrosion: human skin model test
• TG 432 In vitro 3T3 NRU photo toxicity test
• TG 437 Bovine corneal opacity and permeability test
method for identifying ocular corrosives and severe irritants
• TG 438 Isolated chicken eye test method for identifying
ocular corrosives and severe irritants
• TG 439 In vitro skin irritation: reconstructed human
epidermis (RhE) test method

28. 2. Reduction in the number of animals and stress of the
laboratory animals
• TG 420 Acute oral toxicity—fixed dose procedure
• TG 423 Acute oral toxicity—acute toxic class method
• TG 425 Acute oral toxicity—up-and-down procedure
• TG 429 Skin sensitization—local lymph node assay
• TG 436 Acute inhalation toxicity—acute toxic class
method

29. Animal Models
• Animal Testing alternative (s)
• In vitro (method, model,
technique)
• Non-animal alternative (s)
Cell Culture
Tissue Culture
Organ Culture
• Single-cell Organisms
Invertebrates
• Fish
• Simulator (s)
• Mannequin (s)
• Mathematical Model (s)
• Computer-interfaced Interactive
Digital
Image Libraries
Virtual Surgery
Virtual Reality
• Computer simulation (s)
Computer program (s)
Computer Software
Computer Aided
Instruction
• Physiological Simulation (s)
Videodisc (s)
Video display
ALTERNATIVE METHOD TO ANIMAL TOXICITY TESTING

30. The use of animals can be further subdivided according to the
degree of suffering
•Minor animal suffering: Observing animals in behavioural studies,
single blood sampling, immunization without adjutants, etc.
•Moderate animal suffering: Repeated blood sampling, recovery
from general anaesthesia, etc.
• Severe animal suffering: LD50% test, starvation vaccine potency
tests, etc.

31. 1. Cell culture and tissue engineering
I. Skin corrosion and skin irritation
II. Skin absorption
III.Phototoxicity
2. Human-based
I. Skin irritation
II. Pyrogenicity
III.Modular immune in vitro construct
3. Computer simulation
4. Medical imaging
5. Microfluidic Chip
5. Fungal model for mammalian drug metabolism
6. Future alternatives
I. Organs on a chip
II. Human toxome

32. TISSUE CULTURE
• In vitro cultivation of organs, tissues & cells at defined
temperature
using an incubator & supplemented with a medium containing cell
nutrients & growth factors is collectively known as tissue culture
• Different types of cell grown in culture includes connective tissue
elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue
(liver, breast, skin, kidney) and many different types of tumor cells.
Common cell lines
Human cell lines
• -MCF-7 breast cancer
• HL 60 Leukemia
• HEK-293 Human embryonic kidney
• HeLa Henrietta lacks

33. Primate cell lines
• Vero African green monkey kidney epithelial cells
• Cos-7 African green monkey kidney cells
• And others such as CHO from hamster, sf9 & sf21 from insect
cells
• Skin irritation & skin corrosion refers to localized topical
exposure of the skin to a substance
• EpiDerm , Mattek , EpiSkin and Skin Ethic RHE are derived from
human skin cells which cultured to Produce human skin model
• In 2010 august OECD published test guidelines 439 : for irritant
chemicals

34. Skin absorption test
The test substance is applied to the surface of an excised
skin sample in a diffusion cell. Skin from human or animal sources
can be used. Although viable skin is preferred, non-viable skin can
also be used.
Phototoxicity
Phototoxicity is a rash, swelling or inflammation, like a
severe sunburn. the 3T3 Neutral Red Uptake (NRU) Phototoxicity
Test,.
HeLa cells
An immortal cell line used in oncological research derived
from cervical cancer cells

35. ENDOTHELIAL CELLS :
study of natural anticoagulation novel in vitro EC culture
system allows for the screening of endothelial protectants effect of
substances this model also mimic xenotransplantation.
Simulation of stroke related Damage in cultured human nerve
cells
• 2 types of cells : Ntera2 and NT2
• focuses on neuronal cell biology , suitable for screening
neuroprotective drugs
• It has potential to replace lab.animal in basic stroke research
Permanent fish cell culture as imp tool in ecotoxicology
PLHC-1 and RTG-2 cell culture for ecotoxicological tests ,
screening

36. parasite cyst in cultured cells :
N.caninum oocyst which enables generate all stages of this parasite
Render animal model reductant
Aggregating brain cell cultures :
these are 3 dimensional primary cell culture ,study of neuro-
degenerative drugs
Study of angiogenesis : heart of rat n mouse . For screening ,
Mouse kupffer call line : to study diseased condition with microbes,
liver transplantation , tumours , drug delivery

37. II. In-silico methods in Drug Discovery
A. Molecular docking
B. Virtual High through put screening.
C. QSAR (Quantitative structure-activity relationship)
D. Pharmacophore mapping
A. Molecular Docking RL
• Docking is the computational determination of binding affinity
between molecules (protein structure and ligand).
• Given a protein and a ligand find out the binding free energy of
the complex formed by docking them.

38. B. Virtual High through put screening.
1. Receptor based methods-make use of the structure of the target
protein.
2. Ligand based methods-based on the known inhibitors
1. Receptor based methods
•Uses the 3D structure of the target receptor to search for the
potential candidate compounds that can modulate the target function.
•These involve molecular docking of each compound in the chemical
database into the binding site of the target and predicting the
electrostatic fit between them.
•The compounds are ranked using an appropriate scoring function
such that the scores correlate with the binding affinity.
•Receptor based method has been successfully applied in many
targets

39. 2. Ligand based strategy
• In the absence of the structural information of the target, ligand
based method make use of the information provided by known
inhibitors for the target receptor.
• Structures similar to the known inhibitors are identified from
chemical databases by variety of methods,
• Some of the methods widely used are similarity and substructure
searching, pharmacophore matching or 3D shape matching.
• Numerous successful applications of ligand based methods

40. C. QSAR
• QSAR is statistical approach that attempts to relate physical and
chemical properties of molecules to their biological activities.
• Various descriptors like molecular weight, number of rotatable
bonds LogP etc. are commonly used.
• Many QSAR approaches are in practice based on the data
dimensions.
• It ranges from 1D QSAR to 6D QSAR

41. D. Pharmacophore mapping
• It is a 3D description of a pharmacophore, developed by
specifying the nature of the key pharmacophoric features and the
3D distance map among all the key features.
• A Pharmacophore map can be generated by superposition of
active compounds to identify their common features.
• Based on the pharmacophore map either de novodesign or 3D
database searching can be carried out.